HPMA copolymer-1,5-diazaanthraquinone conjugates as novel anticancer therapeutics

1,5-diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC50 for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate>397 microM drug-equiv.) than the free drug (the IC50 for amino-DAQ was 12.6 and 2.8 microM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.     

Development of HPMA copolymer–anticancer conjugates: Clinical experience and lessons learnt

The concept of polymer–drug conjugates was proposed more than 30 years ago, and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate of doxorubicin covalently bound to the polymer backbone by a Gly-Phe-Leu-Gly peptidyl linker (FCE28068) became the first synthetic polymer-based anticancer conjugate to enter clinical trial in 1994. This conjugate arose from rational design attempting to capitalise on passive tumour targeting by the enhanced permeability and retention effect and, at the cellular level, lysosomotropic drug delivery to improve therapeutic index. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA as a new polymer platform. Subsequent Phase I/II trials have investigated an HPMA copolymer-based conjugate containing a doxorubicin and additionally galactose as a targeting moiety to promote liver targeting (FCE28069), and also HPMA copolymer conjugates of paclitaxel (PNU 166945), camptothecin (PNU 166148) and two platinates (AP5280 and AP5346- ProLindac™). The preclinical and clinical observations made in these, and clinical studies with other polymer conjugates, should shape the development of next generation anticancer polymer therapeutics.     

HPMA copolymer–aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10^{? 8} M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [³H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.     

Anticancer carrier-linked prodrugs in clinical trials

Coupling of low molecular weight anticancer drugs to antibodies, serum proteins or polymers through a cleavable linker has been an effective method for improving the therapeutic index of cytotoxic established agents. Modern drug–antibody conjugates that have recently entered clinical trials have primarily used highly potent drugs such as calicheamicin or maytansins. Gemtuzumab ozogamicin, a conjugate of calicheamicin and an anti-CD33 humanized antibody, is the first drug–antibody conjugate to receive market approval. Drug conjugates that have undergone clinical assessment include N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates with doxorubicin, camptothecin, paclitaxel and Pt(II) complexes, poly(ethylene glycol) conjugates with camptothecin and paclitaxel, polyglutamate conjugates with paclitaxel and camptothecin, a methotrexate–albumin conjugate and an albumin-binding doxorubicin prodrug. This review summarizes the Phase I – III studies that have been performed with these macromolecular prodrugs.     

Biorecognition of HPMA Copolymer-Adriamycin Conjugates by Lymphocytes Mediated by Synthetic Receptor Binding Epitopes

Purpose. The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl) metha-crylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. Methods. HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC₅₀ doses determined. Results. Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. Conclusions. The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.     

Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck

Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 μM) the targeted conjugate showed significantly higher uptake (p≤0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p≤0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p≤0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p≤0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.     

Water-soluble HPMA copolymer—prostaglandin E1 conjugates containing a cathepsin K sensitive spacer

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E₁ (PGE₁) delivery system was designed, synthesized and characterized. PGE₁ was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo–initiated free radical copolymerization of HPMA, PGE₁-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the d-aspartic acid octapeptide targeting moieties. Incubation of the PGE₁-containing macromonomer and HPMA copolymer-PGE₁ conjugates with cathepsin K resulted in release of unmodified PGE₁. The rate of release depended on the composition of the conjugate. The higher the PGE₁ content in the conjugate, the slower the PGE₁ release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE₁ conjugates have a potential in the treatment of osteoporosis and other bone diseases.     

HPMA copolymer-aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer-AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM-Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 x 10(- 8) M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2-0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [(3)H]androstenedione both AGM and HPMA copolymer-GFLG-AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.     

HPMA Copolymer-Anticancer Drug-OV-TL-TL16 Antibody Conjugates. 1. Influence of the Method of Synthesis on the Biding Affinity to OVCAR-3 Ovarian Carcinoma Cells In Vitro

The influence of different methods of binding the OV-TL16 antibody and its Fab′ fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer—drug (adriamycin {ADR} or meso chlorin e₆ mono(N-2-aminoethylamide) {Mce₆} conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer—drug (ADR or Mce₆) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling the HPMA copolymer—Mce₆ conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab′ fragment of the OV-TL16 antibody demonstrated a more homogeneous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physicochemical methods where employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce₆) and the spectral properties of Mce₆ were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with changes in antibody conformation.     

Starlike vs. Classic Macromolecular Prodrugs: Two Different Antibody-Targeted HPMA Copolymers of Doxorubicin Studied in Vitro and in Vivo as Potential Anticancer Drugs

Purpose. Two different monoclonal antibody-targeted HPMA copolymer-doxorubicin conjugates, classic and starlike, were synthesized to be used for site-specific cancer therapy. The anti-mouse Thy-1.2 (IgG3) and two anti-human CD71/A (IgG1) and CD71/B (IgG2a) monoclonal antibodies were used as targeting structures. Methods. Their binding and cytotoxic activity in vitro, body distribution, and anticancer activity in vivo were evaluated. Results. The results of flow cytometric analysis showed comparable binding of classic and starlike conjugates to the target cells. The in vitro cytotoxic effect was 10-fold higher if cancer cells were exposed to the starlike conjugate compared to the classic one. Biodistribution studies showed that the starlike conjugate remained in a relatively high concentration in blood, whereas the classic conjugate was found in a 6.5-times lower amount. In contrast to the low antitumor activity of free doxorubicin and nontargeted HPMA copolymer-doxorubicin conjugate, both anti-Thy-1.2 targeted conjugates (classic and starlike) cured all mice bearing T-cell lymphoma EL4. On the other hand, starlike conjugates containing anti-CD71/A or anti-CD71/B monoclonals as targeting structures were more effective against human colorectal cancer SW 620 than the classic one. Conclusions. We have shown that the starlike conjugates are more effective systems for targeted drug delivery and cancer treatment than classic conjugates.     

Gamma Scintigraphy of a 123I-Labelled N-(2-Hydroxypropyl)Methacrylamide Copolymer-Doxorubicin Conjugate Containing Galactosamine Following Intravenous Administration to Nude Mice Bearing Hepatic Human Colon Carcinoma

Abstract

Polymer drug conjugates composed of N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer covalently bound, doxorubicin, and containing additionally galactosamine to facilitate hepatocyte-specific targeting (HPMA copolymer-dox-gal), were synthesised to contain a small amount (～1 mol%) of the monomer methacryloyltyrosinamide to permit radioiodination with [¹²³I]iodide. After intravenous administration to both normal mice and nude mice bearing hepatic human colon carcinoma, the biodistribution of the conjugate was monitored using the gamma camera, and also by dissection analysis. Efficient liver accumulation of HPMA copolymer-dox-gal was seen in the gamma camera images within 20 min, both in normal and rumour-bearing animals. Quantitatively liver uptake was ～40% dose administered/g liver. Images of the tumour-bearing animals showed clearly a much lower accumulation of HPMA copolymer-dox-gal in the colon carcinoma deposits within the liver (3-9% dose/g tumour), and this lack of uptake was verified by ex vivo imaging of the tumour-containing liver and also by dissection analysis. It can be concluded that ¹²³I-labelled HPMA copolymer conjugates offer great potential as effective imaging agents and can be used for future non-invasive clinical studies. This nuclear imaging method will enable optimisation of the dosing schedule by identification of doses of HPMA copolymer-dox-gal that display receptor saturation (and hence diminished targeting efficiency). In addition this conjugate can provide negative images of liver-associated tumour deposits that do not express the asialoglycoprotein receptor.     

Targetable HPMA Copolymer–Aminohexylgeldanamycin Conjugates for Prostate Cancer Therapy

Purpose  This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. Materials and Methods  HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to α_vβ₃ integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. Results  HPMA copolymers containing AH-GDM and RGDfK showed active binding to the α_vβ₃ integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer–AH-GDM–RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. Conclusion  Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer–AH-GDM–RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.     

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

Abstract

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.     

Antibody-targeted Polymer–doxorubicin Conjugates with pH-controlled Activation

The paper is dealing with the synthesis and properties of new non-targeted or antibody-targeted polymer drug conjugates, bearing doxorubicin (DOX) attached via a spacer susceptible to pH-controlled hydrolysis (hydrazone conjugates), designed as anticancer drugs facilitating site-specific therapy. These conjugates are stable in a pH 7.4 buffer, modeling conditions during transport in the body, but release DOX and activate it inside target cells as a result of pH changes when going from outside to inside the cells. Conjugates containing an antibody directed against T lymphocytes bind effectively and specifically T cell lymphoma EL 4 cells. Cytotoxicity of the hydrazone conjugates is higher than that of classic conjugates, depending on the detailed structure of the polymer, the spacer between the drug and polymer carrier and method of antibody conjugation. Cytotoxicity of some of the conjugates is comparable even with that of the free drug. In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the conjugates containing DOX was enhanced with long-term survivors (T-cell lymphoma EL 4, C57BL/6 mice) in comparison with much less effective free DOX or a classic P(N-(2-hydroxypropyl)methacrylamide)HPMA–DOX conjugate (already clinically tested).     

Comparison of active and passive targeting of doxorubicin for somatostatin receptor 2 positive tumor models by octreotide-modified HPMA copolymer-doxorubicin conjugates

Somatostatin receptor 2 (SSTR2), specifically over-expressed on many tumor cells, is a potential receipt for active targeting in cancer therapy. In the present study, octreotide (Oct), which had high affinity to SSTR2, was attached to N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric system to enhance the antitumor efficiency of the anticancer drug doxorubicin (DOX). Two kinds of cell lines (HepG2 and A549), which overexpress SSTR2, were chosen as cell models. Compared with non-modified conjugates, Oct-modified conjugates exhibited superior cytotoxicity and intracellular uptake on both HepG2 and A549 cell lines. This might be due to the mechanism of receptor-mediated endocytosis. Subsequently, the in vivo biodistribution and antitumor activity evaluations showed that Oct modification significantly improved the tumor accumulation and antitumor efficacy of HPMA copolymer conjugates in SSTR2 over-expressed Kunming mice bearing H₂₂ tumor xenografts. In summary, Oct-modified HPMA polymer-DOX conjugates might be a promising system for the treatment of SSTR2 over-expressed cancers.     

Polyacetal-diethylstilboestrol: A Polymeric Drug Designed for pH-triggered Activation

Those polymer anticancer-drug conjugates currently undergoing clinical evaluation have a tripartite structure; a water-soluble polymer, an anticancer agent and a pendant linker. To simplify the construct it would be attractive to develop anticancer polymer therapeutics that contain the bioactive agent as an integral part of the polymer backbone. The aim of this study was to utilise the reaction between a divinyl ethers and diols, to synthesise polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumours would then trigger drug release eliminating the need for a biodegradable linker. A tert-polymerisation approach was used to incorporate non-steroidal oestrogen diethylstilboestrol (DES) into the mainchain of water-soluble polyacetals synthesised using as co-monomer PEG of M_w 2900 or 3400 g/mol. When PEG₂₉₀₀ was used the resultant polymer had a M_w of 18,900 g/mol, a M_w/M_n of 1.9 and a DES loading 4.3 wt.%. With PEG₃₄₀₀ the polymer M_w was 43,000 g/mol, M_w/M_n=1.8 and it had a DES loading 4.7 wt.%. ¹H-NMR confirmed the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25–1.3(d) and 4.7–4.8(q) ppm and from DES at 1.55–1.6(d) and 5.4–5.5(q) ppm. These were consistent with the acetal signals observed for the non-water-soluble co-polymer DES: tri(ethylene glycol) divinyl ether (TEGDVE) (1:2, M_w=6859 g/mol, M_w/M_n=1.3). When evaluated in vitro, the DES-polyacetal displayed greater cytotoxicity than DES against human and murine tumour cell lines (IC₅₀=48 and 420 μg/ml against MCF-7 human breast cancer cells and IC₅₀=97 and 560 μg/ml against B16F10 murine melanoma cells, respectively). These polymers showed no significant haemolysis at concentrations up to 20 mg/ml confirming suitability for further in vivo evaluation. An enhanced rate of hydrolytic degradation of the polymer backbone was seen at pH 5.5, (65% trans-DES released in 96 h), compared to pH 7.4 (4% trans-DES released in 96 h). These bioresponsive DES-polyacetals tert-polymers are the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release of a drug incorporated into the polymer mainchain. Their in vitro characteristics suggest further in vivo evaluation is warranted.     

Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing doxorubicin designed in the late 1970s/early 1980s as anticancer polymer therapeutics were the first synthetic polymer-based anticancer conjugates to enter clinical trial beginning in 1994. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA copolymers as a new polymer platform in this setting. Subsequent Phase I/II trials have investigated conjugates containing paclitaxel (PNU 166945), camptothecin (PNU 166148) (both failed in clinical trials underlining the importance of rational design), and most recently HPMA-copolymer platinates (AP5280 and then AP5346-ProLindac^TM) entered Phase II clinical development. There are a growing array of second generation HPMA copolymer-based systems involving combination therapy, incorporating putative targeting ligands, having an ever more complex architecture, and both drug and protein conjugates are being proposed as novel treatments for diseases other than cancer. Despite their promise, and the success of polymeric drugs and polymer-protein conjugates, no polymer-drug conjugate (HPMA copolymer-based or otherwise) has yet entered routine clinical use. It is timely to reflect on the progress made over the last 30 years, the relative merits of HPMA copolymers as a platform compared to other polymeric carriers, and comment on their future potential as polymer-based nanomedicines into the 21st century in comparison with the many alternative strategies now emerging for creation of nanopharmaceuticals.     

Targeted Delivery of Doxorubicin by HPMA Copolymer-Hyaluronan Bioconjugates

Purpose. Overexpression of hyaluronan (HA) receptors on cancer cells results in enhanced endocytotic uptake of the drug conjugate. An N-(2-hydroxypropyl)methacrylamide (HPMA)-HA polymeric drug delivery system was used for targeted delivery of doxorubicin to cancer cells. Methods. HA-doxorubicin (DOX) bioconjugates (HA-DOX), and HPMA copolymer-DOX conjugates containing HA as a side chain (HPMA-HA-DOX) were synthesized. The cytotoxicity of the polymer-drug conjugate was evaluated via in vitro cell culture. The internalization of the conjugate was visualized by fluorescence microscopy. Results. Cytotoxicity of HPMA-HA-DOX targeted bioconjugate was higher against human breast cancer (HBL-100), ovarian cancer (SKOV-3), and colon cancer (HCT-116) cells when compared to the non-targeted HPMA-DOX conjugate. Fluorescence confocal microscopy revealed that the targeted HPMA-HA-DOX conjugates were internalized more efficiently by cancer cells relative to the non-targeted HPMA-DOX conjugate. Both HPMA-DOX and HPMA-HA-DOX showed minimal cytotoxicity toward mouse fibroblast NIH 3T3 cells. The internalization of polymer conjugates was correlated with their cytotoxicity. Conclusions. Selective delivery of anti-cancer agents to cancer cells was achieved by biochemical targeting. The HA-modified HPMA copolymer showed improved toxicity due to receptor-mediated uptake of the macromolecular drug.     

Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e₆ monoethylenediamine (Mce₆) conjugates was explored to aid in the design of second-generation PDT delivery systems. Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce₆ was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce₆, P-NLS(FITC)-GG-Mce₆) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce₆, P-NLS(FITC)-GFLG-Mce₆). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC₅₀), cell associated drug concentration (CAD) and confocal microscopy. Results. P-GFLG-Mce₆ possessed enhanced chemotherapeutic activity compared to P-GG-Mce₆ indicating enzymatically released Mce₆ was more active than copolymer-bound Mce₆. Lysosomes appeared less sensitive to photodamage as observed by a higher IC₅₀. Nuclear-directed HPMA copolymer-Mce₆ conjugates (P-NLS(FITC)-GG-Mce₆ P-NLS(FITC)-GFLG-Mce₆) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce₆ conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce₆) or amino groups (P-NH₂-GG-Mce₆) also displayed increased chemotherapeutic activity. Conclusions. Nuclear delivery was observed for P-NLS(FITC)-GG-Mce₆ and P-NLS(FITC)-GFLG-Mce₆ indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.     

Biodegradable multiblock poly(N-2-hydroxypropyl)methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment

The synthesis, characterization, and in vitro evaluation of a combination delivery of multiblock poly(N-2-hydroxypropyl)methacrylamide (HPMA), gemcitabine (GEM) and paclitaxel (PTX) conjugates is described in this study. Multiblock copolymer conjugates of a large molecular weight (M_w > 200 kDa) were studied and compared to traditional, small molecular weight (M_w < 45 kDa) conjugates. Stability of the conjugates in different pH was assessed, and their cytotoxicity in combination toward A2780 human ovarian cancer cells was evaluated by combination index analysis. Treatment duration (4 and 72 h) and sequence of addition were explored. In addition, an HPMA copolymer conjugate with both GEM and PTX in the side chains was evaluated in a similar manner and compared to a physical mixture of individual conjugates. Conjugates with narrow molecular weight distribution (M_w/M_n < 1.1) were obtained via RAFT polymerization, and drug loadings of between 5.5 and 9.2 wt% were achieved. Conjugates demonstrated moderate stability with less than 65% release over 24 h at pH 7.4, and near complete drug release in the presence of the lysosomal enzyme cathepsin B in 3 h. In combination, the cytotoxic effects of a mixture of the conjugates were primarily additive. Synergistic effects were observed when A2780 human ovarian cancer cells were treated simultaneously for 4 h with multiblock conjugates (CI < 0.7). When both GEM and PTX were conjugated to the same copolymer backbone, moderate antagonism (CI 1.3–1.6) was observed. These results demonstrate that multiblock HPMA copolymer–GEM and –PTX conjugates, when delivered as a mixture of individual agents, are promising for the treatment of ovarian cancer.